The role of protein kinases in regulating metabolism in lens is being addressed by studying: 1) the protein kinases, and 2) the endogenous proteins that serve as substrates for the lens protein kinases. The focus of this study is on the purification of the protein kinases and the characterization of the phosphorylation of four endogenous substrates by cAMP-dependent protein kinases. The phosphorylated proteins are Alpha-crystallin and 26K and 19K intrinsic membrane proteins. Comparison of the amino acid compositions of the 26K and 19K proteins suggest they are closely related. Detailed structural studies are in progress to determine how similar they are. Compounds that are thought to regulate the function of the 26K protein in vivo modulate the phosphorylation of the protein in vitro. Oxidative changes of lens proteins are thought to occur with aging and to contribute to the development of cataracts. The goals of this project are to determine: 1) the extent of oxidative modification of crystallins and metabolic enzymes in both normal and cataractous lenses; 2) the nature of the modifications and the mechanisms leading to the changes; and 3) the effect of the modifications on proteolysis of these proteins. Bovine and human lenses have been used for these studies. The carbonyl content of lens protein has been used as an indiction of the extent of the oxidative modification. The carbonyl content is determined by reactivity with 2,4-DNPH. These studies have demonstrated in normal lenses here is a low but significant increase in carbonyl content as a function of age up to 100 years. Some types of cataracts and brunescent lenses have significantly increased carbonyl content. Noncataractous lenses obained from diabetic individuals have levels varying from normal to significantly elevated carbonyl levels. In vitro mixed-function oxidase systems are being used to study the oxidative modification of metabolic enzymes and crystallin, and effects on proteolytic degradation. Experiments are in progress to identify the hydrazones.